Subcellular structures are unresolvable by classical optical techniques as such structures are smaller than the resolution limit, as described by Abbe, Beiträge zur Theorie des Mikroskops and der mikroskopischen Wahrnehmung, Arch. Mikroskop. Anat. 9, 413-18 (1873). Super-resolved fluorescence microscopy techniques have been developed to overcome this long-standing far-field resolution barrier in recent years, as described by Hell, et al., Far-field optical nanoscopy, Science 316, 1153-58 (2007), and can be divided into two major approaches. One approach relies on a nonlinear optical response to reduce the width of the point spread function either directly or in post-acquisition image synthesis. The best developed example of such a technique is stimulated-emission-depletion fluorescence microscopy (STED) in which a second laser depletes excited fluorophores that are farthest away from the focal spot, described by Hell, et al., Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy, Opt. Lett. 19, 780-82 (1994).
A second approach circumvents the usual resolution limits by trans-forming imaging into a mapping problem. While imaging is a problem of estimating the components of a vector in an infinite-dimensional Hilbert space, and is limited by bandwidth, mapping is a series of problems each requiring an estimate of two or three components of a vector in a two- or three-dimensional Hilbert space and limited by signal-to-noise ratio (SNR). This reduction in dimensionality of the problem is achieved by limiting the number of active fluorescent probes in the focal region to one. Switchable fluorescent probes provide such control, enabling separation of individual probes in the time domain from spatially overlapping fluorescent images. For each activated probe the position is calculated by finding the center of the imaged spot. A complete image is subsequently built by acquiring a series of subimages. High-precision mapping of probes was independently implemented as PALM (photo activated localization microscopy, described by Betzig, et al., Imaging Intracellular Fluorescent Proteins at Nanometer Resolution, Science, 313, 1642-45 (2006)), STORM (Rust, et al., ISub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM), Nat. Methods, 3, 793-95 (2006)), and FPALM (Hess, et al., Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy, Biophys. J., 91, 4258-72 (2006)). The location of the individual probes can be estimated with a higher precision than the diffraction limit The sequential localization of the fluorescent probes, subsequently, builds up the final map over time. The localization precision is proportional to ∝ 1/√{square root over (N)}, where N is the number of detected photons. Thus, increased resolution requires longer measurements.
As a consequence of the fact that spatial mapping precision depends on the isolation of emission events in time, the major limiting factor of PALM/STORM methods is the intrinsic trade-off between spatial and temporal resolution, making them slower and less suitable than conventional microscopy for dynamic samples. While spatial resolution may be improved by measuring for a longer period, this leads to a reduced temporal resolution. Applications are therefore mainly limited to static or slowly-changing samples.
Strand-like tissue such as microtubules have become a benchmark structure for gauging spatial resolution. Including fluorescent probes with spectrally distinct emission spectra has allowed for parallel acquisition and improves imaging times, as discussed by Gunewardene, et al., Superresolution Imaging of Multiple Fluorescent Proteins with Highly Overlapping Emission Spectra in Living Cells Biophys. J., 101, 1522-28 (2011), incorporated herein by reference. To date the number of differently colored dyes being imaged simultaneously has been limited to four, due to technological challenges discussed by Testa, et al., Multicolor Fluorescence Nanoscopy in Fixed and Living Cells by Exciting Conventional Fluorophores with a Single Wavelength, Biophys. J., 99, 2686-94 (2010). As a result, resolution in PALM/STORM approaches has reached a practical limit dictated by the intrinsic properties of the process as well as by available technology.